Ubiquitin ligases regulate the degradation of the majority of cellular proteins. The largest category of ubiquitin ligases is the family of Cullin-RING ubiquitin ligases (CRLs). CRLs regulate many dynamic cellular processes including the cell cycle, transcription, signal transduction, and development. CRLs are multisubunit complexes that contain a cullin, which forms a rigid scaffold for the assembly of the complex. CRLs require the conjugation of a ubiquitin-like peptide, Nedd8, to the cullin for full activity. Cullins that lack Nedd8 can be bound and sequestered by the inhibitor CAND1. Neddylation and deneddylation events are linked to a cycle of cullin sequestration and CRL complex assembly in a proposed CRL activation cycle. Many aspects of CRL activation are not well understood, including how the association of cullins with CAND1 is regulated. In C. elegans, CAND-1 is an important negative regulator of cullin neddylation, and cullin neddylation levels are significantly increased in cand-1 mutants. CAND-1 is not essential for major CRL functions, but does promote CRL activity in vivo. This proposal employs genetic approaches to uncover how the inhibitory binding of CAND-1 to cullins is regulated and to identify novel regulators of the CRL activation cycle. A genome-wide RNAi screen will be used to identify cand-1 enhancers;and cand-1 suppressors that have previously been isolated from chemical mutagenesis screens will be cloned. Enhancer and suppressor genes will be analyzed molecularly and genetically to determine how they regulate CAND-1 function and CRL activity. Preliminary screens have identified enhancers of the cand-1 mutant that mediate post-translational modification of proteins. We will test the hypothesis that post-translational modifications of cullins regulate their interaction with CAND-1. We have used tandem mass spectrometry to identify post- translational modifications for the cullins CUL-2 and CUL-4. The functional significance of the post-translational modifications will be assessed through the in vivo analysis of cullins with site-directed changes in the modification sites. The proposed genetic and biochemical experiments will further our understanding of the regulatory pathways that control one of the most important classes of ubiquitin ligases. Aberrant regulation of CRLs is implicated in cancer progression, and therefore the insights obtained from this study will have relevance for understanding the genesis of this disease. Notes on changes to Abstract Section: We have already identified cand-1 suppressors, and the abstract has been altered to reflect this by eliminating the screen for new cand-1 suppressor genes. We have also now identified post-translational modifications (PTMs) for the cullins CUL-2 and CUL-4 using an affinity purification/tandem mass spectrometry approach. The abstract has been modified to eliminate the previously proposed screen to identify cullin PTMs.